Dioxins are normally formed due to incomplete combustion processes and belong to a group of robust and mostly toxic chemical substances known as persistent organic pollutants. Among them, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is considered to be the most toxic species and classed as a known carcinogen by the World Health Organization (WHO). The toxicity of the other dioxin species or mixtures of dioxins is normally described in relation to TCDD.
Traditionally, analytical chemical processes, such as chromatographic and mass spectral technologies, are employed to determine the dioxin content in samples. However, these processes are cost-ineffective and time-consuming and, more importantly, the results obtained thereby are not directly correlated to the toxicity of dioxin. All these disadvantages make the analytical chemical processes unsuitable for high throughput screening.
In 1995, the biological effects of dioxins were reported to be mediated by the aryl hydrocarbon receptor (AHR) pathway (Fernandersalguero, et al., Science 268: 722-726, 1995). AHR is a resident protein found in the cytoplasm, which forms a heteromer with two molecules of heat shock protein 90 (HSP90). Dioxin molecules can easily penetrate across the plasma membrane and bind to the heteromer, causing the heteromer to undergo transformation that dissociates AHR from HSP90. The free form of AHR is active to be translocated into the nucleus where it binds to an AHR nuclear translocator (ARNT) protein. The AHR-ARNT complex is proved to be a transcription activator that binds to certain regulatory sequences in DNA, including the so-called dioxin responsive element (DRE), and triggers the expression of downstream effectors. Given the fact that AHR dissociates from HSP90 and then associates with ARNT upon binding to dioxin, a broad variety of bioassays were established in 1990's. For example, Wheelock et al. provided a method of detecting dioxin-like compounds in a test sample (U.S. Pat. No. 5,529,899 filed on Jul. 27, 1993 and issued on Jun. 25, 1996). In this method, the test sample was contacted with a heteromer formed from an inactive AHR. If dioxin-like compounds are present in the test sample, they will bind to the AHR causing it to dissociate from the heteromer as a complex containing active AHR bound to a dioxin-like compound ligand. Then, Wheelock et al. further provided a method of detecting dioxin-like compounds using ARNT to optimize AHR transformation (U.S. Pat. No. 6,127,136 filed on Feb. 14, 2006 and issued on Oct. 3, 2000). Okuyama et al. provided a study on enzyme-linked immunoabsorbent assay (ELISA) using a monoclonal anti-dioxin antibody with a sensitivity at nanomolar level (Okuyama, et al., Anal. Chem. 76: 1948-1956, 2004). However, the above-mentioned methods are dissatisfied because of low sensitivity.
Furthermore, in vitro bioassays based on fluorescence resonance energy transfer (FRET) technique have been developed, which involve assessment of the interactions between AHR and its binding partners to determine the dioxin content in test samples (see, for example, Lin, et al., J. Biomed. Sci. 15: 833-840, 2008; and Lin, et al., Chin. Biosci. 50(1): 12-25, 2007). In this study, a fluorescence resonance energy transfer (FRET)-based dioxin-detection bioassay was established, wherein AHR and ARNT fused-cyan fluorescent protein (CFP) and -yellow fluorescent protein (YFP) constructed were transiently co-transfected into rat hepatoma cell line, H4IIEC3 cells. The results showed that dioxin treatments upregulated FRET signals in the transfected cells in a dose-dependent manner. In spite of the high sensitivity and specificity achieved by these assays, the use of cell-based systems for detecting dioxin-like compounds is less desirable in view of the cumbersome in manipulating living cells and the inherent variability of whole cells.
Recently, a cell-free assay system was provided in WO 2005/100991, where recombinant AHR and ANRT in their partially purified forms were prophetically subjected to FRET analysis. However, no empirical data were shown in this regard.
Therefore, there exists a need for cost-effective and high-throughput methods and cell-free systems to enable rapid and quantitative detection of a dioxin-like compound with high sensitivity.